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TRANSCRIPT
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Pharmaceuticals 2011, 4, 992-1018; doi:10.3390/ph4070992
PharmaceuticalsISSN 1424-8247
www.mdpi.com/journal/pharmaceuticals Review
Methylenedioxymethamphetamine (MDMA, 'Ecstasy'):
Neurodegeneration versus Neuromodulation
Elena Puerta and Norberto Aguirre *
Department of Pharmacology, School of Pharmacy, University of Navarra, Irunlarrea 1, 31008
Pamplona, Spain; E-Mail: [email protected] (E.P.)
* Author to whom correspondence should be addressed; E-Mail: [email protected];
Tel.: +00-34-948-425-600 Ext. 6550; Fax: +00-34-948-425-649.
Received: 22 April 2011; in revised form: 15 June 2011 / Accepted: 4 July 2011 /
Published: 5 July 2011
Abstract: The amphetamine analogue 3,4-methylenedioxymethamphetamine (MDMA,
‘ecstasy’) is widely abused as a recreational drug due to its unique psychological effects.Of interest, MDMA causes long-lasting deficits in neurochemical and histological markers
of the serotonergic neurons in the brain of different animal species. Such deficits include
the decline in the activity of tryptophan hydroxylase in parallel with the loss of 5-HT and
its main metabolite 5-hydoxyindoleacetic acid (5-HIAA) along with a lower binding of
specific ligands to the 5-HT transporters (SERT). Of concern, reduced 5-HIAA levels in
the CSF and SERT density have also been reported in human ecstasy users, what has been
interpreted to reflect the loss of serotonergic fibers and terminals. The neurotoxic potential
of MDMA has been questioned in recent years based on studies that failed to show the loss
of the SERT protein by western blot or the lack of reactive astrogliosis after MDMAexposure. In addition, MDMA produces a long-lasting down-regulation of SERT gene
expression; which, on the whole, has been used to invoke neuromodulatory mechanisms as
an explanation to MDMA-induced 5-HT deficits. While decreased protein levels do not
necessarily reflect neurodegeneration, the opposite is also true, that is, neuroregulatory
mechanisms do not preclude the existence of 5-HT terminal degeneration.
Keywords: 3,4-methylenedioxymethamphetamine (MDMA, ‘ecstasy’); 5-hydroxy-
tryptamine (5-HT, serotonin); neurotoxicity
OPEN ACCESS
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1. Introduction
3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is a phenylpropanolamine with
structural similarities to both amphetamine and mescaline (Figure 1). Ecstasy has come to symbolize
one of the largest youth subcultures of the late 20th century and has established itself as one of themost popular recreational drugs of abuse [1,2]. The appeal of MDMA is related to its unique profile of
psychotropic actions, which includes amphetamine-like stimulant effects, coupled with feelings of
increased emotional sensitivity and closeness to others [3-9]. Based on this, Nichols [10] postulated
that MDMA represents a novel class of compound, in that it possesses unique effects, which means
that it cannot be classified as either a hallucinogen or a psychostimulant. Nichols [10] proposed the
term ‘‘Entactogen’’ to describe the effects of MDMA and related compounds. This definition is based
on the claimed ability of MDMA to allow therapists and patients to access and deal with repressed
painful emotional issues [11]. Due to these unique psychological effects, although never marketed for
this purpose, MDMA was used in the 70s and 80s by U.S. psychotherapists [4]. Nevertheless, concernover its increasing abuse and its possible neurotoxicity in humans led to the assignment of MDMA as a
Schedule I agent by the U.S. Drug Enforcement Agency in 1985 [12]. Since then, there have been
ongoing controversies regarding whether MDMA is medically useful as an adjunct in psychotherapy [13-17]
and whether the neurotoxic findings seen after single or repeated high doses of MDMA in
experimental animals are relevant for humans [18-23].
Figure 1. Chemical structures of MDMA and related compounds.
2. Pharmacology
As an in-depth examination of the preclinical pharmacology of MDMA is beyond the scope of this
mini-review, only a brief summary will be detailed here (for more in-depth reviews, see [24-27]).
Preclinical data gathered from research in laboratory animals, namely in rats, indicate that upon
systemic administration, MDMA affects peripheral and central nervous system (CNS) functions by
acting mainly on monoaminergic systems. Thus, initial studies showed that MDMA and its main
metabolite 3,4-methylenedioxyamphetamine stimulate efflux of [3H]5-hydroxytryptamine (5-HT) [28]
(Figure 1) and [3H]dopamine from preloaded synaptosomes [29,30]. Subsequent reports, using in vivo
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microdialysis, demonstrated that MDMA increases extracellular 5-HT, dopamine and noradrenaline
levels in multiple brain regions with effects on 5-HT being greater in magnitude [31-34]. MDMA also
enhances the release of acetylcholine, an effect that appears to be secondary to the activation of
serotonergic, dopaminergic and/or histaminergic receptors [35]. The evaluation of monoamine release
after MDMA intake has not been studied in humans or non-human primates, but several studies
suggest that the interaction of MDMA with the 5-HT carrier and a subsequent release of 5-HT could be
responsible for most of the physiological and psychological responses to MDMA in humans [5-8,36].
Available evidence also indicates that acute release of dopamine caused by MDMA mediates its
reinforcing effects although non-monoaminergic effects may also be involved. It has also been shown
that repeated MDMA induces behavioral sensitization and cross-sensitization to cocaine, MDMA
substitutes for cocaine in a drug discrimination task, and that MDMA pre-exposure facilitates cocaine
conditioned place preference and intravenous self-administration [37-41]. Despite these findings, a
general consensus of the animal literature is that MDMA is a low-efficacy reinforcer when comparedto self-administration of other drugs of abuse and does not possess the same addictive potential as
stimulants like cocaine or methamphetamine [37]. In a similar fashion, the dependence potential of
MDMA is much weaker than that reported for other drugs of abuse (e.g. opioids, alcohol). While some
people report problems controlling and concern about their use, the notable lack of case reports of
severe withdrawal syndromes in the literature suggests that physical symptoms play a more limited
role than psychological ones [42]. Of interest, diminished subjective effects over repeated uses have
often been reported by human ecstasy users [43], an effect that has been replicated in
self-administration experiments with MDMA conducted in rhesus monkeys over an 18-month period
of contingent drug exposure [39].
3. Toxicology
It is worth noting that MDMA-related medical complications have risen more than 20-fold in recent
years in the U.S.A. and Europe, consistent with increasing popularity of the drug [44-46]. Serious
adverse effects of MDMA intoxication include cardiac arrhythmias, hypertension, hyperthermia,
serotonin (5-HT) syndrome, hyponatremia, liver complications, seizures, coma, and death [45].
However, considering the widespread use of MDMA, fatal intoxications remain rare events [2].
Further, accumulating evidence also indicates that long-term MDMA abuse is associated with
cognitive impairments and mood disturbances, which can last for months after cessation of drug
intake [47-54].
In animals, MDMA can cause long-lasting changes in neurochemical and histological markers of
serotonergic function in brains of rats [18,19,55], primates [56-57] and, possibly, humans [58]. Such
effect is evidenced by the decline in the activity of tryptophan hydroxylase [59]; a decrease in the
content of 5-HT and its main metabolite 5-hydoxyindoleacetic acid (5-HIAA) [18,19,22,60,61]; a
lower density of [3H]paroxetine-labelled 5-HT transporters (SERT) [20,62] along with the loss of
SERT protein in several regions of the brain [63,64]; and long-term altered responses to 5-HT agonists
or 5-HT releasing drugs in rats, non-human primates and MDMA users [65-69]. This constellation offindings, coupled with neuroanatomic observations using different techniques such as
immunohistochemistry [63,70-72] and silver impregnation methods [73], strongly suggest that MDMA
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Importantly, many of the putative confounds of former clinical studies on the effects of MDMA in
the serotonergic neurotransmitter system of abusers were carefully controlled in a recent study carried
out by Kish and co-workers [101]. These authors using a magnetic resonance image for positron
emission tomography image co-registration and structural analyses confirmed a caudorostral gradient
of cortical SERT binding loss with occipital cortex most severely affected in ecstasy abusers in
comparison to control subjects. The SERT binding loss was not related to structural changes or partial
volume effect, use of other stimulant drugs, blood testosterone or oestradiol levels, major SERT gene
promoter polymorphisms, gender, psychiatric status, or self-reported hyperthermia or tolerance.
Furthermore, the ecstasy group, although ‘grossly behaviorally normal’, reported altered mood and
demonstrated generally modest deficits on some tests of attention, executive function and memory
associated with SERT decrease. The direct measurement of major brain 5-HT markers in human
ecstasy users was also accomplished, for the first time, in a single post-mortem brain of a high-dose
user [102]. The authors showed marked decreased levels of all 5-HT neuron markers: 5-HT and5-HIAA content and protein concentrations of its rate limiting biosynthetic enzyme, tryptophan
hydroxylase and of SERT [102]. These studies, therefore, appear to confirm the initial interpretation
that serotonergic neurotransmission is subject to long lasting and subtle alterations following MDMA
abuse in humans.
4. Interspecies Dose Scaling: Allometric versus Effect Scaling
To assess the potential risks to human health arising from chemical exposures such as MDMA, it is
frequently necessary to rely on findings from controlled animal experiments. A major point of
controversy relates to the relevance of MDMA doses administered to rats when compared to dosestaken by humans [37]. The neurotoxic effects of MDMA in animals are dependent upon the number
and size of doses given, the route of administration, the species receiving it, and the ambient
temperature in which it is received [24,26]. Thus, MDMA regimens that produce 5-HT depletions in
rats and non-human primates involve administration of single or multiple injections of 7.5-20 mg/kg or
5 mg/kg respectively, whereas the typical amount of MDMA taken by human users is 1-3 mg/kg.
Allometry is the measurement of the shape of an organism as it increases in size, and its principles
have been used to extrapolate doses from one species to another. Allometric interspecies scaling is
based on the assumption that there are anatomical, physiological, and biochemical similarities among
animals, which can be described by mathematical models. It is now a well-established fact that many
physiological processes and organ sizes exhibit a power-law relationship with the body weight of
species. This relationship is the scientific basis of allometry. However, while some physiological,
anatomical, and biochemical characteristics vary in a systematic manner, others do not. For example,
(i) bioavailability and extent of binding to plasma proteins may be species-specific [103]; (ii) partition
coefficients can also exhibit large differences across species [104]; (iii) qualitative differences between
the human brain and those for other mammalian species (brain:body weight ratio and increased brain
surface area due to folding in higher mammalian species) complicates the interspecies extrapolation for
delivery to the brain [105]; (iv) allometric scaling yields poor predictions in humans for low clearancedrugs eliminated primarily by the mixed function oxidase system [106]. Differences among animal
species regarding other pharmacokinetic parameters such as drug absorption (bioavailability),
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variations in metabolic enzymes and their activities or the phenomenon of nonlinear kinetics are other
factor that should be taken into account at the time of using the interspecies scaling to extrapolate toxic
MDMA doses between animals and humans [reviewed by 28,107].
Due to the limitations of allometric scaling, other authors have proposed to use the effect scaling, as
an alternative strategy for matching equivalent doses of MDMA in rats or non-human primates and
humans [25, 108]. The principle behind interspecies effect scaling is that drug doses which induce
identical effects (behavioral, physiological, neurochemical, etc.) in two different species may be
considered equivalent, and therefore directly compared across these particular species, suggesting that
the method of effect scaling can account for differences in ADME processes across species. Baumann
et al. [25] compared the doses of MDMA capable of producing similar neurobiological effects in rats
and humans (“effect scaling”). They concluded that there is no justification for using allometric
interspecies scaling to adjust rats and human doses since MDMA produces comparable psychological
effects at similar doses (~1-2 mg/kg) in both species. Of notice, these findings contrast with otherfunctional observations. Thus, a standard dose of MDMA (100 mg; 1.5 mg/kg p.o.) given to humans in
a control setting increases oral temperature 0.6ºC [109] and a similar rise is seen after a fourfold higher
dose (5 mg/kg i.p.) administered to rats [110]; which, importantly, represents the rat/human dose ratio
calculated by using the allometric interspecies scaling.
Which one of these two methods (allometric vs. effect scalling), if any, best predicts the dosage
regimen that might result neurotoxic in humans based on neurotoxic studies carried out in rats or non-
human primates awaits further studies. For instance, the effect scaling method predicts that the dose of
1.7 mg/kg of MDMA used by Vollenweider et al. [9] in healthy humans would be completely safe
since repeated treatment in rats with behaviorally relevant doses of MDMA (1.5 mg/kg i.p.) does notcause any sign of long-term 5-HT deficits [25]. Similar findings were reported by
Fantegrossi et al. [39], who found no significant differences in VMAT binding or tissue levels of
5-HT, 5-HIAA, dopamine or DOPAC in rhesus monkeys that self-administered MDMA over
an 18-month period. By contrast, the allometric scaling method predicts the opposite because a dose of
1.7 mg/kg given to healthy human volunteers (or self-administered by ecstasy users) is in the range of
those reported to be neurotoxic in rats and non-human primates [111-113]. Noteworthy, in this latter
work Drs. McCann and Ricaurte [113] remarked that the principles of interspecies scaling do not apply
when the metabolic disposition in particular animal species involves the formation of a unique
neurotoxic metabolite. So far, it is still unclear which are the mechanisms underlying MDMA-inducedlong-term 5-HT deficits. Some authors have cautiously suggested that peak plasma concentration of
MDMA (Cmax) is the pharmacokinetic parameter that best correlates with the degree of 5-HT loss both
in rats and non-human primates [93,114]; however, other authors have proposed the metabolism of
MDMA into toxic metabolite(s) as responsible for its neurotoxic effects (Figure 2), see [26,115] for
further discussion.
As suggested elsewhere, the optimal strategy for relating MDMA data from animals to humans
might be to compare pharmacokinetic parameters across species [28,107,116,117]. This is exactly
what it was done in two recent studies in which the metabolic disposition of MDMA in laboratory
animals (rats or squirrel monkeys) and humans was compared [116,117]. In the first study by Baumann
and co-workers [116], rats received a low, but pharmacologically active, dose of 2 mg/kg or a 5-fold
higher dose of MDMA via intraperitoneal, subcutaneous or oral routes. Neither dose given via any of
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the three routes of administration resulted in long-term depletions of 5-HT in the cortex or striatum of
the rat. Of interest, the lower dose, given orally or intraperitoneally, yielded an MDMA Cmax of
approximately 46 and 200 ng/mL, respectively. Two conclusions can be withdrawn from these data: (i)
the effect scaling method cannot be used to appropriately estimate exposure to MDMA in relation to
dose administered; (ii) in rats, an MDMA Cmax in the range of those reported in humans who receive
1.3 to 1.7 mg/kg orally in a control setting [118-122], which represents a typical recreational single
dose [49], does not result in long-lasting 5-HT deficits. An interesting conclusion withdrawn from the
second study by Mueller et al. [117] refers to the impossibility of producing identical exposures in
squirrel monkeys and humans despite using the interspecies dose scaling due to big differences in T 1/2
of MDMA between humans and monkeys. Thus, estimated equivalent doses through the use of
interspecies dose scaling yields similar AUC values but not Cmax values, being the opposite also true,
that is, non-equivalent dosages produce comparable Cmax values but different AUC values. Obviously,
this also applies to the rat [116]. However, as discussed by Mueller et al. [117] in the case of MDMAthe most important pharmacokinetic parameter to better predict the neurotoxicity induced by this
amphetamine derivative awaits to be defined, mainly because we still do not know what is causing the
long-lasting 5-HT deficits described in rats, monkeys or humans.
5. Can We Prevent the Long-Term 5-HT Deficits Caused by MDMA in Rats?
In the following section we will briefly summarize the different paradigms that have been shown to
reverse the serotonergic deficits observed after MDMA administration. All the studies presented refer
to experiments carried out in rats since these type of experiments have not been conducted in
non-human primates.
5.1. Core Body Temperature and 5-HT Deficits
Many in vivo studies show the close relationship between hyperthermia and neurotoxicity
engendered by MDMA [123-126]. In this regard, any pharmacological or non-pharmacological
manipulations capable of preventing the acute hyperthermic response caused by MDMA also attenuate
or reverse the serotonergic deficits induced by the drug.
Keeping animals at low ambient temperatures during MDMA administrations [123,126] or surgical
treatments such as hypophysectomy or thyrophysectomy [127] prevent both the acute hyperthermiaand the long-term deficits observed in the serotonergic system after its administration.
Additionally, many compounds, that were initially reported to be neuroprotective in rats, have been
confirmed to afford protection because of an indirect effect by lowering body temperature. That is the
case of some N -methyl-D-aspartic acid (NMDA) glutamate receptor antagonists (MK-801, CGS 19755,
NBQX) [128]; the 5-HT2A receptor antagonist, ketanserin [129]; the γ-aminobutyric acid (GABAA)
agonist, clomethiazole [130]; the dopamine synthesis inhibitor, α-methyl- p-tyrosine (AMPT) [129]; the
anesthetic pentobarbitone [131] and the glucose transport inhibitor 2-deoxy-D-glucose [62]. In all these
cases, when the core body temperature of rats was kept elevated, the neuroprotective effect of these
drugs was lost. Of interest, the converse also occurs; any manipulation known to potentiate MDMA-induced hyperthermia, such as small increases in ambient temperature [124,132,133] or caffeine
administration [134] enhance the serotonergic deficits.
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5.2. Inhibition of ROS Formation
Several studies suggest the involvement of oxidative stress in the mechanism of MDMA
neurotoxicity. For instance, the administration of free radical scavengers and antioxidants such as
phenylbutylnitrone, ascorbate and α-lipoic acid attenuates the serotonergic deficits induced bythe drug [135-137]. Supporting these evidences is the fact that enhancing neuronal anti-oxidative
defense by acetyl-L-carnitine prevents the decrease of 5-HT levels in several regions of the rat brain
after MDMA administration [138]. In addition, the antioxidant and glutathione precursor
N -acetylcysteine also prevented in vitro thioether MDMA metabolite-induced neuronal death [139].
5.3. Dopamine and Its Metabolism by MAO-B
It had been proposed that one source of free radicals observed after MDMA administration is the
metabolism of dopamine by monoamine oxidase-B (MAO-B) to produce H2O2 that is reduced by ironto produce hydroxyl radicals and a subsequent terminal degeneration [140-143]. This suggestion is
supported by the fact that MDMA elicits dopamine release [144] and the dopamine precursor
L-3,4-dihydroxyphenylalanine (DOPA) exacerbates MDMA-induced 5-HT depletions in the rat [145].
Conversely, inhibition of dopamine synthesis and blockade of its uptake by mazindol attenuates
MDMA-induced long-term depletion of brain 5-HT content [146,147]. Moreover, inhibition of
MAO-B, either by pharmacological agents (L-deprenyl also termed selegiline) or antisense
oligonucleotide targeted at MAO-B, was shown to be protective [140,141,148,149]. Importantly,
a recent study has shown that MAO-A inhibition by clorgyline had no protective effect on MDMA-
induced toxicity but the opposite [150]. The concomitant use of MAO-A inhibitors and MDMA iscounter indicated because of the resulting severe synergic toxicity.
It should also be noted, however, that rats depleted of vesicular and cytoplasmic dopamine stores by
means of previous treatment with reserpine and α-methyl- p-tyrosine (AMPT) showed no deficits of 5-
HT after MDMA. Importantly, these animals did not turned hyperthermic but hypothermic and when
this effect was averted by raising ambient temperature, the 5-HT neuroprotective effects of reserpine
and AMPT were no longer apparent, suggesting that dopamine per se is not essential for the expression
of MDMA-induced 5-HT neurotoxicity [125].
5.4. Inhibition of Nitric Oxide Synthase and Peroxynitrite Formation
Darvesh et al. [151] demonstrated that the systemic administration of MDMA to rats significantly
increased the formation of nitric oxide and the nitrotyrosine in the striatum. These results support the
conclusion that nitrogen reactive species (formed by the reaction of NO with O2•− radicals) could also
be involved in MDMA damage. So, systemic administration of the nitric oxide synthase (NOS)
inhibitors N -nitro-L-arginine (L- NOARG) or S -methyl-L-thiocitrulline inhibits brain NOS activity and
provides protection against MDMA-induced indole depletion without attenuating the acute
hyperthermia induced by this amphetamine analog [151,152]. Of interest, some in vitro studies have
also demonstrated that the non-selective NOS inhibitor, N -ω-nitro-L-arginine [153] and specificinhibitors of the inducible and neuronal nitric oxide synthase (NOS) [154] partially prevented MDMA
neurotoxicity, further suggesting the involvement of reactive nitrogen species in the toxic effect.
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5.5. Inhibition of SERT-Mediated Transport
The involvement of the 5-HT transporter (SERT) in the long-term effects induced by MDMA has
been evidenced by many studies. Thus, the 5-HT uptake inhibitor fluoxetine given up to seven days
before, at the time or up to six hours after MDMA, prevents both the acute increase in hydroxyl radicalformation which follows MDMA [149] and the long-lasting serotonergic deficits without interfering
with MDMA-induced hyperthermia [30, 55, 132,156]. This neuroprotective effect might be due to the
blockade of the entry of a toxic metabolite of MDMA into the 5-HT nerve terminal [156]. However,
taking into account the studies suggesting the involvement of MDMA metabolism in its neurotoxicity
[126,133] and the fact that fluoxetine is a potent enzymatic inhibitor, the protection afforded by the
SERT inhibitor may also be a result of a pharmacokinetic interaction between fluoxetine and
MDMA [157].
5.6. Pharmacological Preconditioning
Recent studies have reported that intermittent administration of MDMA to adolescent and adult rats
resulted in tolerance to subsequent 5-HT-depleting regimen of the drug, as well as to the long-term
reduction in SERT immunoreactivity [158-160]. Noteworthy this neuroprotective effect was
independent of any alteration in MDMA pharmacokinetics or MDMA-induced hyperthermia.
In general, the neuroprotective effects of prior intermittent MDMA exposure can be considered an
example of ‘‘preconditioning,’’ a phenomenon whereby low doses or brief exposures to noxious
insults protect the brain and other tissues from future insults [161,162].
Additionally, Puerta et al. [163] have recently shown that sildenafil, given shortly before MDMA,also prevented the long-term 5-HT deficits caused by MDMA in rats by a preconditioning-like
mechanism. Of interest, minoxidil also afforded protection against MDMA-induced 5-HT depletion by
a similar mechanism [164] including the activation of ATP-sensitive potassium (KATP) channels, an
essential initiator of preconditioning in different models [165-169]. Moreover, 3-nitropropionic acid,
given 24 h before a toxic MDMA treatment, completely prevented 5-HT depletions by increasing NO
production [170]; which is also involved in various preconditioning paradigms [171,172]. In all these
studies, the neuroprotective effect was independent of any alteration in the hyperthermia induced
by MDMA.
Thus, pharmacological preconditioning is also a valid strategy to prevent the 5-HT depletionscaused by MDMA. Although the mechanisms underlying this protective effect remain to be
determined, attenuation of ROS production after development of preconditioning during lethal
challenges is thought to be one of the major end effectors in this process [173-175].
5.7. 5-HT Precursors
Treatment of rats with 5-HT precursors, tryptophan or 5-hydroxytryptophan, was shown to
attenuate MDMA-induced serotonergic deficits as measured by [3H]-paroxetine binding and 5-HT
content in the striatum, hippocampus, and frontal cortex of the rat brain [176]. These authors suggestedthat 5-HT depleted terminals are more vulnerable to the toxic effects of MDMA, and so, 5-HT
precursors would protect 5-HT terminals by replenishing the vesicular stores.
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Figure 2. Proposed mechanisms underlying MDMA-induced serotonergic neurotoxicity.
Toxic MDMA metabolites, increased tyrosine levels and dopamine metabolism act in
concert inside the serotonergic terminals to promote oxidative stress and final terminal loss.
Other factors, e.g. hyperthermia, by increasing MDMA metabolism, may contribute to
MDMA-induced serotonergic deficits. Adapted from [115].
6. Neuroadaptation vs. Neurotoxicity
During the last few years, several studies have questioned the neurotoxic potential of MDMA to
5-HT terminals. Such conclusions were based on results from western blot studies of the SERT protein
showing no change in SERT protein abundance despite large decreases of 5-HT concentrations and
[3H]paroxetine binding after, what up to that date, had been considered a neurotoxic MDMA treatment
in rats [25,177,178]. However, results of western blot studies indicate that the 50 kDa band previouslythought to correspond to the SERT protein [178] does not exhibit the known relative regional
distribution of brain SERT, is resistant to the well established 5-HT neurotoxicant 5,7-dihydroxy-
tryptamine, and is present in SERT-KO animals. It appears, therefore, that the failure of previous
studies to demonstrate the loss of SERT protein by Western blot relies on the fact that the band
corresponding to SERT protein was misidentified, probably due to the lack of positive and negative
controls used by other authors [63,179]. Recent studies have shown a significant decreased abundance
of a ~65 kDa band, corresponding to the SERT protein, in MDMA-treated rats [64,159,179,180],
further supporting the conclusions reached by the initial study carried out by Xie and co-workers [63].
The previously reported studies showing no loss of the SERT protein band after MDMA, is not theonly argument some authors put forward to question the 5-HT neurotoxic potential of MDMA.
Reactive astrogliosis defined as an increase in the number and size of astrocytes can occur as a result
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of numerous acute or chronic stresses compromising brain homeostasis. Glial fibrillary acidic protein
(GFAP) is the major protein constituent of astroglial intermediate filaments and has been used as a
marker to detect neuronal degeneration [181]. There are few studies reporting the use of this marker to
examine MDMA-induced damage and most of them reported no changes in GFAP expression after
several treatment regimens with MDMA known to deplete central 5-HT concentrations
[177,178,181,182]. This was further supported by the lack of an MDMA mediated effect on markers of
microglial activation such as heat shock protein expression and peripheral benzodiazepine receptor
binding [177,178,182]. These and other findings continue to raise doubts among some investigators as
to whether MDMA “serotonergic neurotoxicity” involves distal axotomy or alternatively a long-lasting
downregulation of 5-HT synthesis and SERT expression by the serotonergic neurons. It should be
noted however, that besides the study by Aguirre et al. [137], at least two other studies have
documented increases in GFAP expression after MDMA although at three but not seven days after
MDMA [183] or after a very high dose of the drug [184]. Of interest, data from different studies using5,7-DHT are also not consistent, with some studies revealing increased GFAP expression after
neurotoxic regimens [177], whereas others report similar findings in old but not in young animals
[185] or no increase at all [186-188]. It has been suggested that a lack of GFAP expression increase
may be due to an insufficiently strong signal after 5-HT neuronal degeneration [186]; that the use of
this parameter for detecting degeneration of serotonergic terminals may have limitations [63] or,
alternatively, that changes in GFAP expression are transient and can only be detected shortly after
neuronal injury caused by MDMA [183].
In a recent study by Wang et al. [189], a group of rats was treated with a neurotoxic dosage regimen
of MDMA and two weeks later these same animals received an injection of the 5-HT precursor5-hydroxytryptophan. Interestingly, 5-HT levels in the brain of MDMA-pretreated rats were close to
those of control animals. Because SERT or TPH activity are not necessary for the conversion of
5-hydroxytryptophan into 5-HT these authors interpreted these data as a proof of concept for the
integrity of 5-HT terminals. Accordingly, they suggested that MDMA causes lasting neuroadaptative
changes in 5-HT neurons rather than 5-HT terminal loss. Later on, a different report showed that a
“neurotoxic” dosage regimen of MDMA in rats did not affect VMAT-2 protein expression in the
hippocampus despite producing large reductions in SERT levels [64]. This scenario is not consistent
with the idea of MDMA being neurotoxic to 5-HT terminals in rats but it rather supports the
neuroregulatory hypothesis proposed by Wang et al. [189]. The study of Wang et al. [189] alsoevinced the difficulty in determining the effects of MDMA when measuring endpoints such as 5-HT
content, SERT protein expression, binding or function. These measures are all indirect and subject to
regulations. For instance, it is well known that MDMA inhibits the activity [190] and abundance of
tryptophan hydroxylase [191] at least two weeks after MDMA. Because tryptophan hydroxylase is the
rate-limiting enzyme for 5-HT synthesis, it is likely that decreases in this enzyme restrict the extent of
5-HT production, thus reducing the levels of this neurotransmitter regardless of whether or not axonal
damage has occurred. In this same line, SERT binding is also subject to various regulatory processes,
and changes in SERT binding levels cannot, as a single line of evidence, connote the loss of 5-HT
terminals. For example, chronic treatment of rats with 5-HT selective reuptake inhibitors (SSRIs) leads
to a marked loss of SERT binding and function comparable to MDMA [192], although, such changes
are transient and not long-lasting like those produced by MDMA. Further support for the above
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contention comes from two recent studies. In the first report, Kivell and co-workers [193] showed that
MDMA causes a redistribution of SERT from the cell surface to intracellular vesicles. These authors
therefore suggested that the loss of SERT from the cell surface upon acute exposure to MDMA might
contribute to the decreased SERT function seen in rats exposed to MDMA. However we should be
cautious at the time of interpreting these latter results, since this is expected to be an acute effect of
MDMA and cannot account for the very long-lasting reductions reported in rats, non-human primates
or ecstasy users. In the second study, Biezonski and Meyer [64] reported a profound down-regulation
of SERT gene expression accompanied by a significant reduction in the expression of the
VMAT-2 gene in the dorsal and median raphe nuclei of rats two weeks after a high dosage regimen of
MDMA. The authors indicated that decreased protein levels liable to regulation (e.g. SERT or VMAT-
2 seen after MDMA) do not necessarily reflect neurodegeneration. While this is true and could explain
the loss of SERT protein found after MDMA in different animal species, the fact is that the opposite is
not less certain. Therefore, a down-regulation of SERT protein expression does not preclude theexistence of 5-HT terminal degeneration. Another unsolved question relates to the duration of the
effects reported by Biezonski and Meyer [64] and later confirmed by Kirilly [194], since SERT mRNA
was found to be below control levels by two and three weeks after MDMA, respectively, while the
effects of MDMA last between 6-12 months in rats [195] and beyond seven years in non-human
primates [56]. Moreover, studies in non-human primates have shown that, over time, there is regrowth
of ascending serotonin axonal projections after MDMA-induced injury but that a normal innervation
pattern is not restored [71].
Wang et al. [189] proposed three possible models for MDMA effects on the serotonergic system:
(1) neurodegeneration, (2) neuroadaptation, and (3) a mixed model involving a significant loss ofserotonergic nerve terminals along with adaptative changes in the remaining terminals. While the new
findings reported by Biezonski and Meyer [64] and Bonkale and Austin [191] are consistent with the
neuroadaptative model, it should be noted that they do not preclude the possibility that MDMA causes
a partial degeneration of serotonergic nerve terminals. In this regard, it has been shown that MDMA
administration does not lead to ultrastructural alteration in the serotonergic dorsal raphe cell bodies and
in their proximal neurites but causes impairment in cortical serotonergic axons. In these, the main
ultrastructural alteration is the destruction of microtubules although a smaller portion of these axons
probably undergoes an irreversible damage [196]. These data support the clinical findings reported by
Kish et al. [101,102] and also the mixed model of neurotoxicity suggested by Wang et al. [189]. It isalso important to consider that the “neurodegeneration” hypothesis can account for all the findings
reported in the scientific literature while the “modulation” hypothesis cannot, at this moment. More
studies are, therefore, warranted.
Finally we would like to mention that the terms “neurotoxicity” and “neurodegeneration” in relation
to amphetamines have been used interchangeably, when the former can occur without the latter. In this
regard, the National Institute of Neurological Disorders and Stroke (NINDS) defines neurotoxicity as
follows: “ Neurotoxicity occurs when the exposure to natural or manmade toxic substances
(neurotoxicants) alters the normal activity of the nervous system. This can eventually disrupt or even
kill neurons, key cells that transmit and process signals in the brain and other parts of the nervous
system. Neurotoxicity can result from exposure to substances used in chemotherapy, radiation
treatment, drug therapies, and organ transplants, as well as exposure to heavy metals such as lead and
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mercury, certain foods and food additives, pesticides, industrial and/or cleaning solvents, cosmetics,
and some naturally occurring substances. Symptoms may appear immediately after exposure or be
delayed. They may include limb weakness or numbness; loss of memory, vision, and/or intellect;
headache; cognitive and behavioral problems; and sexual dysfunction. Individuals with certain
disorders may be especially vulnerable to neurotoxicants”.
As reviewed above, MDMA, at the very least, causes severe deficits in different markers of the
serotonergic neurotransmitter system in rodents, non-human primates and humans [24,26,101,102] and
causes long-lasting cognitive and behavioral problems [48,52,197] and therefore fulfills the definition
of a neurotoxicant compound provided by the NINDS. Whether, it also causes neurodegeneration of
5-HT axons still awaits confirmation.
Acknowledgments
This work was supported by grants from the Ministerio de Ciencia e Innovación (SAF2008-05143-C03-03)to NA.
Conflict of Interest
The authors declare no conflict of interest.
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